Single-Molecule Analysis of Genome Uncoating from Individual Human Rhinovirus Particles, and Modulation by Antiviral Drugs.

Infection of humans by many viruses is typically initiated by the internalization of a single virion in each of a few susceptible cells. Thus, the outcome of the infection process may depend on stochastic single-molecule events. A crucial process for viral infection, and thus a target for developing antiviral drugs, is the uncoating of the viral genome. Here a force spectroscopy procedure using an atomic force microscope is implemented to study uncoating for individual human rhinovirus particles. Application of an increasing mechanical force on a virion led to a high force-induced structural transition that facilitated extrusion of the viral RNA molecule without loss of capsid integrity. Application of force to virions that h ad previously extruded the RNA, or to RNA-free capsids, led to a lower force-induced event associated with capsid disruption. The kinetic parameters are determined for each reaction. The high-force event is a stochastic process governed by a moderate free energy barrier (≈20 kcal mol-1 ), which results in a heterogeneous population of structurally weakened virions in which different fractions of the RNA molecule are externalized. The effects of antiviral compounds or capsid mutation on the kinetics of this reaction reveal a correlation between the reaction rate and virus infectivity.

Electromechanical Photophysics of GFP Packed Inside Viral Protein Cages Probed by Force-Fluorescence Hybrid Single-Molecule Microscopy.

Packing biomolecules inside virus capsids has opened new avenues for the study of molecular function in confined environments. These systems not only mimic the highly crowded conditions in nature, but also allow their manipulation at the nanoscale for technological applications. Here, green fluorescent proteins are packed in virus-like particles derived from P22 bacteriophage procapsids. The authors explore individual virus cages to monitor their emission signal with total internal reflection fluorescence microscopy while simultaneously changing the microenvironment with the stylus of atomic force microscopy. The mechanical and electronic quenching can be decoupled by ≈10% each using insulator and conductive tips, respectively. While with conductive tips the fluorescence quenches and recovers regardless of the structural integrity of the capsid, with the insulator tips quenching only occurs if the green fluorescent proteins remain organized inside the capsid. The electronic quenching is associated with the coupling of the protein fluorescence emission with the tip surface plasmon resonance. In turn, the mechanical quenching is a consequence of the unfolding of the aggregated proteins during the mechanical disruption of the capsid.

Enhancing Visible-Light Photocatalysis via Endohedral Functionalization of Single-Walled Carbon Nanotubes with Organic Dyes.

The encapsulation of an organic dye, 10-phenylphenothiazine (PTH), in the inner cavity of single-walled carbon nanotubes (SWNTs) as a breaking heterogenization strategy is presented. The PTH@oSWNT material was microscopically and spectroscopically characterized, showing intense photoemission when illuminated with visible light at the nanoscale. Thus, PTH@oSWNT was employed as a heterogeneous photocatalyst in single electron transfer dehalogenation reactions under visible light irradiation. The material showed an enhanced photocatalytic activity, achieving turnover numbers as high as 3200, with complete recyclability and stability for more than eight cycles. Computational calculations confirm that electronic communication between both partners is established because, upon illumination, an electron of the excited PTH is transferred from the π system of the molecule to the delocalized π-cloud of the SWNT, thus justifying the enhanced photocatalytic activity.